The objective of the proposed research is to pursue studies on glutaric acidemia type II, a human inborn error of amino and fatty acid oxidation, and on ETF:ubiquinone oxidoreductase (ETF:QO), the protein which is deficient in some patients with the disease and which normally catalyzes electron transfer between electron transfer flavoprotein (ETF) and the mitochondrial respiratory chain. Our investigations on this subject have been ongoing for more than ten years, and are now positioned to substantially improve our understanding of how electron transfer is effected between the flavin and iron-sulfur redox centers of ETF:QO and coenzyme Q (ubiquinone) in the inner mitochondrial membrane, and how mutations of the protein perturb its function and lead to glutaric acidemia type II. Knowledge of how this protein functions is crucial if treatment for the disease by gene transfer approaches, when and if they are developed, is to be successful, information about the gene and its regulation may be important in understanding why ETF:QO deficiency is so frequently associated with severe congenital renal cystic disease. We now have on hand over 95 percent of pork ETF:QO cDNA in five clones in BlueScript, and about 85 percent of the human ETF:QO cDNA in two clones. Specific aims for this funding period are to (a) complete analysis of pork and human ETF:QO cDNA, (b) to use the human probe to define mutations in ETF:QO-deficient GA2 patients, and (c) to develop a structure-function map of ETF:QO, identifying domains which interact with ETF, the flavin and iron-sulfur redox centers, and ubiquinone. Experiments using site-directed mutagenesis of the ETF-QO of Paracoccus denitrificans will be performed to address issues of how changes in amino acid sequence in various domains alter enzyme function.